Horses (Equus caballus) have been considered one of the most significant domesticated animals in human use, including sports, urban and rural transportation and military logistics. Paternity confirmation and breed identification is the basic pre-requisite for rearing specific breeds and maintaining the pedigree records of horses. DNA finger printing has been successfully used for paternity and breed confirmation. Mainly DNA finger printing is done through microsettalite markers for paternity analysis and breed characterization. The aim of this study was to develop and apply a panel of microsettalite markers for paternity analysis and selected horse breeds characterization. Three horse breeds Percheron, Pak Arab and Thoroughbred were selected for this purpose. Sampling of 20 families (Foals, Mare and Stallion) of three selected horse breeds was conducted from Remount Depot Mona. The blood samples was collected in sterilized Falcon tubes each containing lOOjiL EDTA (0.2 mM). DNA was extracted from all blood samples by using inorganic method. The microsatellite markers were selected from ISAG (International Society of Animal Genetics), Indian and TKY recommended panels. The primers were designed for these microsatellite markers using primer3 free ware. All Microsatellite markers used were direpeats. Conditions for successful amplification by PCR (Polymerase Chain Reaction) were optimized and microsatellite markers were grouped into 6 multiplexes (tn and diplex). PCR products of optimized multiplexes were visualized on U V illuminator after gel electrophoresis. DNA amplification was done through PCR by using optimized multiplexes of microsatellite markers for all the samples. Polyacrylarnide Gel Electrophoresis (PAGE) was used for genotyping of amplified DNA samples. Allele sizes of all amplicons were calculated by relative flow method. Alleles for all microsatcilite markers were analyzed statistically by "POPGEN 32 and POWER STAT" software. The investigation revealed average PlC value 0.97, average observed heterozygosity 0.923 1, average observed homozygosity 0.0769, combined power of exclusion (P.E) 0.9999 and combined polymorphic loci percentage for 13 microsettalite markers was 100%. Perchron breed showed genetic identity with Pak Arab and Thoroughbred up to 0.3470 and 0.6157 respectively. Pak Arab exhibited genetic identity with Thoroughbred horse up to 0.6616 where as Perchron breed showed genetic distance with Pak Arab and Thoroughbred up to 1.0585 and 0.4850 respectively. Pak Arab exhibited genetic distances with Thoroughbred up to 0.413 1. Results of analysis were used to describe the new microsettalite markers assay for parentage confirmation and breed characterization of selected breeds (Perchron, Pak Arab and Thoroughbred) horses. 'Ihis panel of microsetallite marker was useful and reliable tool for individual identification and parentage analysis in horses. This microsettalite marker panel can be used on commercial basis in Pakistan.